Methods and compositions for sequencing a nucleic acid
OwnerSuhaib Siddiqi, Philip Buzby +2inventors
InventorsSuhaib Siddiqi, Philip Buzby, Noubar Afeyan, David Liu
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Abstract
The invention provides a family of nucleotide analogs useful in sequencing nucleic acids containing a homopolymer region comprising, for example, two or more base repeats, and to sequencing methods using such nucleotide analogs.
Claims47
A nucleotide analog of Formula I or Formula II:
- wherein,
- B1 and B2 are each independently selected from the group consisting of a purine, a pyrimideine, and analogs thereof;
- R1 and R2 at each occurrence are selected from the group consisting of OH, NH2, F, N3, and H;
- Y is selected from the group consisting of NR′, O, S, CH2, and a bond, wherein R′ is selected from the group consisting of H, alkyl, alkenyl, and alkynyl;
- A is selected from the group consisting of —S—S—, an ester, and an amido group;
- R3 is selected from the group consisting of:
- alkyl, and a bond;
- R4 is selected from the group consisting of alkyl, alkenyl, alkynyl, ether, and a bond;
- R5 is selected from the group consisting of:
- alkyl, alkenyl, and a bond;
- Ar is aryl;
- R6 is selected from the group consisting of:
- R7 is alkyl or a bond;
- R8 is selected from the group consisting of S, alkyl, alkenyl, alkynl, and NR′;
- R9 is selected from the group consisting of NR′, O, S, and —(CH2)m—;
- L is a label;
- X is H or a halogen;
- Z, at each occurrence, independently, is O or S;
- m, at each occurrence, independently is an integer from 0 to 50,
- n, at each occurrence, independently is an integer from 0 to 50, and
- p, at each occurrence, independently is an integer from 0 to 50.
A nucleotide analog represented by:
- wherein B1 and B2 are each independently selected from the group consisting of cytosine, uracil, thymine, adenine, guanine, and analogs thereof;
- PPPO— is
- and
- Z, at each occurrence, independently is O or S.
A method of sequencing a nucleic acid template comprising:
- (a) exposing a nucleic acid template comprising first and second consecutive bases that is hybridized to a primer having a 3′ end to (i) a polymerase which catalyzes nucleotide additions to the primer, and (ii) a labeled nucleotide analog comprising a first nucleotide or a first nucleotide analog covalently bonded through a linker to a blocking group, under conditions to permit the polymerase to add the labeled nucleotide analog to the primer at a position complementary to the first base while preventing another nucleotide or nucleotide analog from being added to the primer at a position complementary to the second base;
- (b) detecting the nucleotide analog added to the primer in step (a);
- (c) removing the blocking nucleotide or blocking nucleotide analog;
- and
- (d) repeating steps (a), (b) and (c) to determine the sequence of the template.
An improved method of sequencing a nucleic acid template containing a homopolymer region using a primer complementary to at least a portion of the template and a polymerase, wherein the improvement comprises performing each cycle of a sequencing reaction in the presence of a labeled nucleotide analog comprising a first nucleotide or first nucleotide analog covalently bonded through a linker to a blocking group under conditions to cause the polymerase to add only a single labeled nucleotide analog to a chain extending from the primer at a position complementary to one base in the homopolymer region.
Dependencies are inferred from the claim text.